High-concentration stem cell production method

ABSTRACT

The present invention relates to a method for preparing stem cells in high concentration. The present invention makes it possible to grow stem cells in an amount sufficient to be clinically usable in a short time, and makes it possible to relatively efficiently enhance the ability of administered stem cells to efficaciously reach target tissue and exhibit an action in a stable fashion and can therefore dramatically increase the efficacy of cell therapy using stem cells.

TECHNICAL FIELD

The present invention relates to a method for preparing a highconcentration of stem cells, and more particularly to a method forgrowing stem cells in high yield sufficient for clinical application.

BACKGROUND ART

Stem cells refer to cells having not only self-replicating ability butalso the ability to differentiate into at least two types of cells, andcan be divided into totipotent stem cells, pluripotent stem cells, andmultipotent stem cells. Totipotent stem cells are cells havingtotipotent properties capable of developing into one perfect individual,and these properties are possessed by cells up to the 8-cell stage afterthe fertilization of an oocyte and a sperm. When these cells areisolated and transplanted into the uterus, they can develop into oneperfect individual. Pluripotent stem cells, which are cells capable ofdeveloping into various cells and tissues derived from the ectodermal,mesodermal and endodermal layers, are derived from an inner cell masslocated inside of blastocysts generated 4-5 days after fertilization.These cells are called “embryonic stem cells” and can differentiate intovarious other tissue cells but not form new living organisms.Multipotent stem cells, which are stem cells capable of differentiatinginto only cells specific to tissues and organs containing these cells,are involved not only in the growth and development of various tissuesand organs in the fetal, neonatal and adult periods but also in themaintenance of homeostasis of adult tissue and the function of inducingregeneration upon tissue damage. Tissue-specific multipotent cells arecollectively called “adult stem cells”.

Adult stem cells are obtained by taking cells from various human organsand developing the cells into stem cells and are characterized in thatthey differentiate into only specific tissues. However, recently,experiments for differentiating adult stem cells into various tissues,including liver cells, were dramatically successful, which comes intospotlight. In particular, efforts have been made in the field ofregenerative medicine for regenerating biological tissues and organs andrecovering their functions that were lost due to illness or accident andthe like by using cells. Methods which are frequently used in this fieldof regenerative medicine comprise the steps of: collecting stem cells,blood-derived mononuclear cells or marrow-derived mononuclear cells froma patient; inducing the proliferation and/or differentiation of thecells by tube culture; and introducing the selected undifferentiated(stem cells and/or progenitor cells) and/or differentiated cells intothe patient's body by transplantation. Accordingly, existing classicalmethods for treating diseases by medication or surgery are expected tobe replaced with cell/tissue replacement therapy which replaces a damagecell, tissue or organ with healthy one, and thus the utility of stemcells will further increase.

Thus, the various functions of stem cells are currently being studied.Particularly, since cell therapy technologies using mesenchymal stemcells started to receive attention, technologies for improvingmesenchymal stem cells isolated from a human body so as to be suitablefor therapeutic purposes have been developed (WO 2006/019357, KoreanPatent No. 0795708, and Korean Patent No. 0818214).

However, a technology related to a method for proliferating the isolatedstem cells in an amount sufficient for clinical application has not yetbeen sufficiently studied.

Accordingly, the present inventors have made many effort to prepare stemcells in large amounts, and have found that, when stem cells arecultured in a medium containing a basal medium and at least twocomponents selected from the group consisting of N-acetyl-L-cysteine(NAC), ascorbic acid, insulin or insulin-like factor, hydrocortisone,basic fibroblast growth factor (bFGF), EGF (epidermal growth factor),and antioxidant, they can be proliferated at a high concentration in ashort period of time, thereby completing the present invention.

The above information disclosed in this Background section is only forenhancement of understanding of the background of the present invention,and therefore it may contain information that does not form the priorart that is already known to a person of ordinary skill in the art.

DISCLOSURE OF INVENTION Technical Problem

It is an object of the present invention to provide a method forpreparing stem cells at a high concentration in a short period of time.

Technical Solution

To achieve the above objects, the present invention provides a methodfor preparing a high concentration of stem cells, the method comprisingof culturing stem cells in a medium containing a basal medium; and atleast two components selected from the group consisting ofN-acetyl-L-cysteine (NAC), ascorbic acid, insulin or insulin-likefactor, hydrocortisone, dexamethasone, bFGF (basic fibroblast growthfactor), heparan sulfate, 2-mercaptoethanol, EGF (epidermal growthfactor), and antioxidant.

Other features and embodiments of the present invention will be moreapparent from the following detailed descriptions and the appendedclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphic diagram showing a cell population doubling levelduring the culture of adipose-derived stem cells obtained from adultmales aged 20, 30, 70 and 80 years in each of media for 4 days.

BEST MODE FOR CARRYING OUT THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Generally, the nomenclatureused herein and the experiment methods are those well known and commonlyemployed in the art.

As used herein, the term “stem cells” refer to cells having not onlyself-replicating ability but also the ability to differentiate into atleast two types of cells. “Adult stem cells” refer to stem cells thatappear either in the stage in which each organ of an embryo is formedafter the developmental process or in the adult stage.

As used herein, the term “mesenchymal stem cells” refers toundifferentiated stem cells that are isolated from human or mammaliantissue and may be derived from various tissues. Particularly, themesenchymal stem cells may be umbilical cord-derived mesenchymal stemcells, umbilical cord blood-derived mesenchymal stem cells, bonemarrow-derived mesenchymal stem cells, adipose-derived mesenchymal stemcells, muscle-derived mesenchymal stem cells, nerve-derived mesenchymalstem cells, skin-derived mesenchymal stem cells, amnion-derivedmesenchymal stem cells, or placenta-derived mesenchymal stem cells.Technology for isolating stem cells from each tissue is already known inthe art.

As used herein, “adipose tissue-derived mesenchymal stem cells” areundifferentiated adult stem cells isolated from adipose tissue and arealso referred to herein as “adipose-derived adult stem cells”, “adiposestem cells”, or “adipose-derived stem cells”. These cells can beobtained according to any conventional method known in the art. A methodfor isolating adipose tissue-derived mesenchymal stem cells may be, forexample, as follows. That is, adipose-derived mesenchymal stem cells canbe isolated by culturing an adipose-containing suspension (inphysiological saline) obtained by liposuction, and then eithercollecting a stem cell layer, attached to a culture container such as aflask, by trypsin treatment, or directly collecting those suspended in asmall amount of physiological saline by rubbing with a scraper.

As used herein, the terms “preparation of a high concentration of stemcells” means that the number of stem cells proliferates in a high yield.As used herein, the terms “high concentration of stem cells” is alsoreferred to as “large number of stem cells”. In an example of thepresent invention, it was confirmed that when stem cells isolated tissueare cultured in a medium according to the present invention, the numberof stem cells obtained is significantly increased. Preferably, when itwas confirmed that when subcultured 3-5 times, the stem cells can beobtained at a concentration of 1×10⁷-5×10⁸ cells/ml.

As used herein, the term “subculture” means that a portion of cellsperiodically passaged by transferring them to a new culture vessel andby replacing the culture medium with a fresh culture for the long-termculture of healthy cells. As the number of cells increases in thelimited space of culture vessel, the cells die naturally due to nutrientdepletion or waste accumulation in a predetermined time. Thus,subculture is used to increase the number of healthy cells. Typically, 1passage means culture by one replacement of medium (culture vessel) orone isolation of cell population. Any subculture method known in the artmay be used without any limitation, but a mechanical or enzymaticisolation method may be preferably performed.

As used herein, the term “mechanical isolation” means that cellaggregates are isolated physically or mechanically. Any mechanicalisolation method known in the art may be used without any limitation,but cells may be preferably isolated by using a blade, a tissue chopper,a needle, pipetting, an EBD (embryoid body divider) or a scrapper.According to a preferred embodiment of the present invention, it wasconfirmed that large-scale proliferation of stem cells can be achievedby subculturing them using the blade or the tissue chopper.

As used herein, the term “enzymatic isolation” means dissociation ofcell aggregates by an enzymatic treatment. Any enzymatic isolationmethod known in the art may be used without any limitation, but cellaggregates may be preferably dissociated by treatment with collagenaseincluding collagenase I, II, III, and IV, accutase, dispase or trypsin,followed by subculturing.

Stem cells can be administered into the body by various routes, forexample, intravenously, intra-arterially or intraperitoneally. Amongsuch administration routes, intravenous administration is preferred,because it enables a disease to be treated in a convenient and safemanner without surgical operation. In order for intravenouslyadministered stem cells to securely reach the target site and to exhibita desired therapeutic effect, various requirements should be satisfied.First, stem cells should be administered at a certain concentration orhigher concentration such that they exhibit a desired therapeutic effectafter they reached the target site. Thus, it is important that stemcells which it is desired to apply clinically are obtained in largeamounts. In addition, stem cells should have a size suitable forintravascular administration such that when administeredintravascularly, these stem cells neither reduce blood flow velocity norform thrombi. Moreover, stem cells should not be disrupted or aggregatedbefore intravascular administration, and should securely reach theirtarget site as single cells without disruption or aggregation even afterintravascular administration.

In view of several requirements as described above, the presentinvention is intended to provide at a certain concentration or higherconcentration of stem cells that are effective clinically. According tothe culture of stem cells by a conventional method, a subculture must beperformed many times to obtain a high yield of stem cells, and thus muchmanpower and time are spent. In particular, since a portion of mediumcomponents necessary for subculture is greatly expensive, it isdisadvantageous in terms of economic efficiency. However, the presentinvention enables stem cells to be prepared in high concentrationsufficient for clinical application.

In one aspect, the present invention is directed to a method forpreparing a high concentration of stem cells, the method comprising astep of culturing stem cells in a medium containing a basal medium andat least two components selected from the group consisting ofN-acetyl-L-cysteine (NAC), ascorbic acid, insulin or insulin-likefactor, hydrocortisone, dexamethasone, bFGF (basic fibroblast growthfactor), heparan sulfate, 2-mercaptoethanol, EGF (epidermal growthfactor), and antioxidant.

Preferably, the stem cells used in the present invention may use adultstem cells, particularly adult stem cells obtained from adipose tissue,or epithelial tissue such as pilar cyst, amnion or the like. Mostpreferably, adipose tissue-derived adult stem cells may be used as thestem cells. Mesenchymal stem cells (MSCs) may be used as the stem cells,particularly adipose tissue-derived mesenchymal stem cells may be usedas the stem cells. The adipose tissue or epithelial tissue is preferablyderived from mammals, more preferably human. In an example of thepresent invention, human adipose tissue-derived mesenchymal stem cells(AdMSCs) were used.

The basal medium used in the present invention refers to a typicalmedium having a simple composition known as being suitable for theculture of stem cells in the art. Examples of the basal medium generallyused to culture the stem cells include MEM (Minimal Essential Medium),DMEM (Dulbecco modified Eagle Medium), RPMI (Roswell Park MemorialInstitute Medium), and K-SFM (Keratinocyte Serum Free Medium). As thebasal medium used in the present invention, any mediums can be usedwithout any limitation as long as they are used in the art. Preferably,the basal medium may be selected from the group consisting of M199/F12(mixture) (GIBCO), MEM-alpha medium (GIBCO), low-concentrationglucose-containing DMEM medium (Welgene), MCDB 131 medium (Welgene),IMEM medium (GIBCO), K-SFM, DMEM/F12 medium, PCM medium, and MSCexpansion medium (Chemicon). Particularly, among them, K-SFM may bepreferably used.

A basal medium that is used to obtain the cultured mesenchymal stemcells may be supplemented with additives known in the art, which promotethe proliferation of mesenchymal stem cells in an undifferentiated statewhile inhibiting the differentiation thereof. Also, the medium maycontain a neutral buffer (such as phosphate and/or high-concentrationbicarbonate) in isotonic solution, and a protein nutrient (e.g., serumsuch as FBS, FCS (fetal calf serum) or horse serum, serum replacement,albumin, or essential or non-essential amino acid such as glutamine orL-glutamine). Furthermore, it may contain lipids (fatty acids,cholesterol, an HDL or LDL extract of serum) and other ingredients foundin most stock media of this kind (such as insulin or transferrin,nucleosides or nucleotides, pyruvate, a sugar source such as glucose,selenium in any ionized form or salt, a glucocorticoid such ashydrocortisone and/or a reducing agent such as β-mercaptoethanol).

Also, for the purpose of preventing cells from adhering to each other,adhering to a container wall, or forming too large clusters, the mediummay advantageously contain an anti-clumping agent, such as one sold byInvitrogen (Cat #0010057AE).

Among them, one or more of the following additional additives mayadvantageously be used:

-   -   stem cell factor (SCF, Steel factor), other ligands or        antibodies that dimerize c-kit, and other activators of the same        signaling pathway    -   ligands for other tyrosine kinase related receptors, such as the        receptor for platelet-derived growth factor (PDGF), macrophage        colony-stimulating factor, Flt-3 ligand and vascular endothelial        growth factor (VEGF)    -   factors that elevate cyclic AMP levels, such as forskolin    -   factors that induce gp130 such as LIF or Oncostatin-M    -   hematopoietic growth factors such as thrombopoietin (TPO)    -   transforming growth factors such as TGFβ1    -   neurotrophins such as CNTF    -   antibiotics such as gentamicin, penicillin or streptomycin.

The medium that is used in the present invention may contain, inaddition to the basal medium, at least two components selected from thegroup consisting of N-acetyl-L-cysteine (NAC), insulin or insulin-likefactor, hydrocortisone, basic fibroblast growth factor (bFGF), andantioxidant.

The medium used in the present invention may contain a basal medium andat least two components selected from the group consisting ofN-acetyl-L-cysteine (NAC), ascorbic acid, insulin or insulin-likefactor, hydrocortisone, dexamethasone, bFGF (basic fibroblast growthfactor), heparan sulfate, 2-mercaptoethanol, EGF (epidermal growthfactor), and antioxidant.

Specifically, the medium may contain insulin-like factor as insulinreplacement, which functions to promote cell growth by enhancing glucosemetabolism and protein metabolism. Particularly, recombinant IGF-1(insulin-like growth factor-1) is preferably used. The preferred contentof insulin-like factor is 10-50 ng/ml. If the content of insulin-likefactor is less than 10 ng/ml, apoptosis will occur, and if the contentis more than 50 ng/ml, it will increase the cytotoxicity and cost of themedium.

The medium may contain basic fibroblast growth factor (bFGF) that caninduce various types of cell proliferation in vivo. Preferably,recombinant bFGF protein is used. The preferred content of bFGF is 1-100ng/ml.

Examples of an antioxidant that may be used in the present inventioninclude selenium, ascorbic acid, vitamin E, catechin, lycopene,β-carotene, coenzyme Q-10, EPA (eicosapentaenoic acid), DHA(docosahexanoic acid) and the like. Preferably, selenium may be used. Inan example of the present invention, selenium was used as anantioxidant. The content of selenium in the medium is preferably 0.5-10ng/ml. If the content of selenium is less than 0.5 ng/ml, the mediumwill be sensitive to oxygen toxicity, and if the content is more than 10ng/ml, it will cause severe cytotoxicity.

The medium that is used in the present invention may additionallycontain a component selected from the group consisting of FBS (fetalbovine serum), calcium and EGF. Epidermal growth factor (EGF) can inducevarious types of cell proliferation in vivo, and recombinant EGF proteinis preferably used. The preferred content of epidermal growth factor is10-50 ng/ml. If the content of epidermal growth factor in the medium isless than 10 ng/ml, it will have no particular effect, and if thecontent is more than 50 ng/ml, it will be toxic to cells.

Preferably, when subcultured 3-5 times, stem cells cultured in themedium according to the present invention are proliferated at aconcentration of 1×10⁷-5×10⁸ cells/ml. In addition, sincefunctional/morphological deformation of cells does not occurs during theincrease in the number of the stem cells by the subculture, the stemcells obtained according to the present invention can be effectivelyapplied to a clinical trial.

In an example of the present invention, adipose-derived mesenchymal stemcells were cultured in the medium of the present invention.Adipose-derived mesenchymal stem cells can be obtained in the followingmanner. First, human adipose tissue obtained from the abdomen byliposuction or the like is isolated and washed with PBS, after which thetissue is cut finely and degraded using DMEM medium containingcollagenase. The degraded tissue is washed with PBS and centrifuged at1000 rpm for 5 minutes. The supernatant is removed, and the pelletremaining at the bottom is washed with PBS, and then centrifuged at 1000rpm for 5 minutes. The resulting cells are filtered through a 100-meshfilter to remove the debris, and then washed with PBS. The cells arecultured overnight in DMEM medium (10% FBS, 2 mM NAC, 0.2 mM ascorbicacid), and then the cells that did not adhere to the bottom of theculture container were washed out with PBS, and the cells aresubcultured while the medium was replaced with K-SFM medium containingNAC, ascorbic acid, calcium, rEGF, insulin, Bfgf, hydrocortisone, andselenium at 2-day intervals, thereby obtaining adipose-derivedmesenchymal stem cells. In addition to this method, any method known inthe art may also be used to obtain mesenchymal stem cells.

EXAMPLES

Hereinafter, the present invention will be described in further detailwith reference to examples. It will be obvious to a person havingordinary skill in the art that these examples are illustrative purposesonly and are not to be construed to limit the scope of the presentinvention.

Example 1 Isolation of Human Adipose Tissue-Derived Mesenchymal StemCells

Adipose tissue isolated from abdominal tissue by liposuction was washedwith PBS and cut finely, after which the tissue was digested in DMEMmedia supplemented with collagenase type 1 (1 mg/ml) at 37° C. for 2hours. The collagenase-treated tissue was washed with PBS andcentrifuged at 1000 rpm for 5 minutes. The supernatant was removed, andthe pellet was washed with PBS and then centrifuged at 1000 rpm for 5minutes. The resulting cells were filtered through a 100-mesh filter toremove debris, after which the cells were washed with PBS and culturedovernight in DMEM medium containing 10% FBS, 2 mM NAC(N-acetyl-L-cysteine) and 0.2 mM ascorbic acid.

Then, non-adherent cells were removed by washing with PBS, and theremaining cells were cultured for 4 passages while the medium wasreplaced with K-SFM (keratinocyte serum free medium) containing 5% FBS,2 mM NAC, 0.2 mM ascorbic acid, 0.09 mM calcium, 5 ng/ml rEGF, 5 μg/mlinsulin, 10 ng bFGF and 74 ng/ml hydrocortisone at 2-day intervals,thereby isolating adipose-derived mesenchymal stem cells.

Example 2 Investigation of the Effect of Medium Components on theAbility of Stem Cells to Proliferate

Medium (i.e., medium 1) containing all of FBS, N-acetyl-L-cysteine(NAC), ascorbic acid, insulin, hydrocortisone, bFGF (basic fibroblastgrowth factor), EGF (epidermal growth factor), and selenium, which areactive ingredients added to the K-SFM medium used in Example 1 and media(i.e., media 2-9) free of at least one of the active ingredients, wereprepared.

Medium composition is as follows:

Medium 1 (M1): K-SFM medium+FBS+NAC+ascorbicacid+insulin+hydrocortisone+bFGF+EGF+selenium

Medium 2 (M2): K-SFM medium+NAC+ascorbicacid+insulin+hydrocortisone+bFGF+EGF+selenium (exclusion of FBS fromingredients of medium 1)

Medium 3 (M3): K-SFM medium+FBS+NAC+ascorbicacid+insulin+hydrocortisone+EGF+selenium (exclusion of bFGF fromingredients of medium 1)

Medium 4 (M4): K-SFM medium+FBS+NAC+ascorbicacid+hydrocortisone+bFGF+EGF+selenium (exclusion of insulin fromingredients of medium 1)

Medium 5 (M5): K-SFM medium+FBS+NAC+ascorbicacid+insulin+bFGF+EGF+selenium (exclusion of hydrocortisone fromingredients of medium 1)

Medium 6 (M6): K-SFM medium+FBS+NAC+ascorbicacid+insulin+hydrocortisone+bFGF+selenium (exclusion of EGF fromingredients of medium 1)

Medium 7 (M7): K-SFMmedium+FBS+NAC+insulin+hydrocortisone+bFGF+EGF+selenium (exclusion ofascorbic acid from ingredients of medium 1)

Medium 8 (M8): K-SFM medium+FBS+ascorbicacid+insulin+hydrocortisone+bFGF+EGF+selenium (exclusion of NAC fromingredients of medium 1)

Medium 9 (M9): K-SFM medium+FBS+NAC+ascorbicacid+insulin+hydrocortisone+bFGF+EGF (exclusion of selenium fromingredients of medium 1)

Medium 10 (M10): K-SFM medium+FBS+NAC+ascorbicacid+insulin+hydrocortisone+selenium (exclusion of bFGF and EGF fromingredients of medium 1).

The adipose-derived stem cells were cultured in the medium.Adipose-derived stem cells obtained from adult males aged 20, 30, 70 and80 years were used as the adipose stem cells. After being cultured ineach of the media for 4 passages, the obtained adipose-derivedmesenchymal stem cells were treated with trypsin, and then the number ofthe cells was measured with a confocal microscope. As can be seen fromTable 1 below, when 1×10⁵ adipose-derived mesenchymal stem cells wereinoculated into the medium at 3 passages and incubated for 4 days, thenumber of cells obtained was listed on a basis of media and days. As aresult, according to the stem cell culture method of the presentinvention, it could be confirmed that when the stem cells are culturedfor 4 days, they could be prepared at a maximum concentration of 2.5×10⁶cells/ml depending on the medium components (see Table 1). In addition,although there is a slight difference in cell population doubling level(CPDL) by age, medium 9 showed the highest cell population doublinglevel (CPDL). In case of adult males aged 20 and 30 years, it could beconfirmed that when the stem cells were inoculated into the medium at aconcentration of 1×10⁵ cells/ml and incubated for 4 days, the number ofthe cells was increased 13-25 folds. In case of adult males aged 70 and80 years, it could be also confirmed that the number of the cells wasincreased 7-15 folds (see FIG. 1). In addition, cells in which mutationoccurred were not confirmed (not shown).

TABLE 1 Investigation of medium composition for preparation of highconcentration of stem cells Post seeding Total Cell Count (× 10⁴/ml) AgeDay M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 20 Day0 10 10 10 10 10 10 10 10 10 10Day1 8 16 15 13 12 16 12 12 8 12 Day2 39 13.5 30.5 38 29.5 33.5 39 3026.5 9.5 Day3 115 11.5 70 110 115 110 110 110 105 11 Day4 230 10.5 135195 180 225 250 230 205 13 30 Day0 10 10 10 10 10 10 10 10 10 10 Day1 1210 14 11 16 27 23 10 26 25 Day2 35 12 24.5 40 40 35.5 33.5 39.5 41 10.5Day3 120 150 0 90 110 95 105 110 125 10.5 Day4 215 10 150 185 130 165230 220 220 13 70 Day0 10 10 10 10 10 10 10 10 10 10 Day1 12 7 16 12 1320 10 13 20 11 Day2 37 9 31 32.5 27.5 29 29.5 30.5 26 13 Day3 85 12 6065 80 86 90 100 95 12 Day4 155 7 85 120 135 105 145 150 155 13.5 80 Day010 10 10 10 10 10 10 10 10 10 Day1 20 10 17 15 16 13 16 13 17 16 Day224.5 11.5 16.5 21 19.5 26 17 24.5 23 10.5 Day3 37 7.5 31 41 37 47 65 5548.5 9 Day4 75 9 55 90 70 70 65 80 65 14.5

INDUSTRIAL APPLICABILITY

According to the present invention, since stem cells can be prepared inan amount sufficient for clinical application even in culture for 3-5passages, the effect of intravascular administration of the stem cellson cell therapy can be significantly increased.

Although the present invention has been described in detail withreference to the specific features, it will be apparent to those skilledin the art that this description is only for a preferred embodiment anddoes not limit the scope of the present invention. Thus, the substantialscope of the present invention will be defined by the appended claimsand equivalents thereof.

1. A method for preparing stem cells at a high concentration of1×10⁷˜5×10⁸ cells/ml, the method comprising culturing stem cells in amedium containing a basal medium; and at least two components selectedfrom the group consisting of N-acetyl-L-cysteine (NAC), ascorbic acid,insulin or insulin-like factor, hydrocortisone, dexamethasone, bFGF(basic fibroblast growth factor), heparan sulfate, 2-mercaptoethanol,EGF (epidermal growth factor), and antioxidant.
 2. The method of claim1, wherein the basal medium is selected from the group consisting ofM199/F12 (mixture) (GIBCO), MEM-alpha medium (GIBCO), low-concentrationglucose-containing DMEM medium (Welgene), MCDB 131 medium (Welgene),IMEM medium (GIBCO), K-SFM, DMEM/F12 medium, PCM medium, and MSCexpansion medium (Chemicon).
 3. The method of claim 1, wherein theantioxidant is selected from the group consisting of selenium, ascorbicacid, vitamin E, catechin, lycopene, β-carotene, coenzyme Q-10, EPA(eicosapentaenoic acid), and DHA (docosahexanoic acid).
 4. The method ofclaim 3, wherein the antioxidant is selenium.
 5. The method of claim 1,wherein the medium additionally contains a component selected from thegroup consisting of FBS (fetal bovine serum), calcium, and EGF.
 6. Themethod of claim 1, wherein the stem cells are adipose tissue-derivedmesenchymal stem cells.